Physical Interaction between Epidermal Growth Factor Receptor and DNA-dependent Protein Kinase in Mammalian Cells*
- From the Cell Growth Regulation Laboratory, Department of Clinical Investigation, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030
Abstract
Binding of extracellular ligands to epidermal growth factor receptors (EGFR) activate signal transduction pathways associated with cell proliferation, and these events are inhibited by monoclonal antibodies against EGFR. Since efficient DNA repair in actively growing cells may require growth factor signaling, it was of interest to explore any linkage between EGFR-mediated signaling and DNA-dependent protein kinase (DNA-PK), an enzyme believed to be involved in repairing double strand breaks and V(D)J recombination. We report that anti-EGFR monoclonal antibodies (mAbs), and not EGFR ligands, trigger a specific early physical interaction between EGFR and a 350-kDa catalytic subunit of DNA or its regulatory heterodimeric complex Ku70/80, in a variety of cell types, both in vivo and in vitro. Inhibition of EGFR signaling by anti-EGFR mAb was accompanied by a reduction in the levels of the DNA-PK and its activity in the nuclear fraction. Confocal imaging revealed that a substantial amount of DNA-PK was co-localized with EGFR in anti-EGFR mAb-treated cells. Anti-EGFR mAb-induced physical interaction between EGFR and DNA-PK or Ku70/80 was dependent on the presence of EGFR, but not on the levels of EGFR. The EGFR associated with DNA-PK or Ku70/80 retains its intrinsic kinase activity. Our findings demonstrate the existence of a novel cellular pathway in mammalian cells that involves physical interactions between EGFR and DNA-PK or Ku70/80 in response to inhibition of EGFR signaling. Our present observations suggest a possible role of EGFR signaling in maintenance of the nuclear levels of DNA-PK, and interference in EGFR signaling may possibly result in the impairment of DNA repair activity in the nuclei in anti-EGFR mAb-treated cells.
Footnotes
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↵* This work was supported in part by American Institute for Cancer Research Grants 94B93 and 96A077, National Institutes of Health Grant CA65746, and new research program funds from The University of Texas M. D. Anderson Cancer Center.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ These authors contributed equally to this study.
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↵§ To whom correspondence should be addressed: Cell Growth Regulation Section, The University of Texas M. D. Anderson Cancer Center (Box 36), 1515 Holcombe Blvd., Houston, TX 77030. E-mail:rkumar{at}notes.mdacc.tmc.edu.
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↵1 The abbreviations used are: EGFR, epidermal growth factor receptor; mAb, monoclonal antibodies; DNA-PK, catalytic subunit of DNA-dependent protein kinase; TGF, transforming growth factor; PI, phosphatidylinositol; PMSF, phenylmethylsulfonyl fluoride; PAGE, polyacrylamide gel electrophoresis; FITC, fluorescein isothiocyanate; TRITC, tetramethylrhodamine B isothiocyanate; immunoprecipitation.
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↵2 M. Mandal, L. Adam, J. Mendelsohn, and R. Kumar, submitted for publication.
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- Received September 22, 1997.
- Revision received November 14, 1997.
- The American Society for Biochemistry and Molecular Biology, Inc.
